Photobiomodulation regulates adult neurogenesis in the hippocampus in a status epilepticus animal model

Status epilepticus (SE) refers to a single seizure that lasts longer than typical seizures or a series of consecutive seizures. The hippocampus, which is vulnerable to the effects of SE, has a critical role in memory storage and retrieval. The trisynaptic loop in the hippocampus connects the substructures thereof, namely the dentate gyrus (DG), CA3, and CA1. In an animal model of SE, abnormal neurogenesis in the DG and aberrant neural network formation result in sequential neural degeneration in CA3 and CA1. Photobiomodulation (PBM) therapy, previously known as low-level laser (light) therapy (LLLT), is a novel therapy for the treatment of various neurological disorders including SE. However, the effects of this novel therapeutic approach on the recovery process are poorly understood. In the present study, we found that PBM transformed SE-induced abnormal neurogenesis to normal neurogenesis. We demonstrated that PBM plays a key role in normal hippocampal neurogenesis by enhancing the migration of maturing granular cells (early neuronal cells) to the GCL, and that normal neurogenesis induced by PBM prevents SE-induced hippocampal neuronal loss in CA1. Thus, PBM is a novel approach to prevent seizure-induced neuronal degeneration, for which light devices may be developed in the future.

Status epilepticus (SE) refers to a single seizure that lasts longer than typical seizures or a series of consecutive seizures. The non-return to baseline status seen in SE causes rapid and widespread neuronal damage. The degree of damage depends on seizure severity and duration. Dysfunctional electrical activity of the central nervous system is associated with neurodegeneration, abnormal neurogenesis in the hippocampus, and behavioral and cognitive deficits 1,2 .
The hippocampus, which is vulnerable to the effects of SE, has a critical role in memory storage and retrieval. The trisynaptic loop in the hippocampus connects the substructures thereof, namely the dentate gyrus (DG), CA3, and CA1. The entorhinal cortex provides input to the DG (synapse 1), which in turn provides input to the CA3 through the mossy fiber pathway (synapse 2). CA3 provides input to the CA1 through the Schaffer collateral pathway (synapse 3). Finally, the CA1 is connected to the entorhinal cortex, thereby completing the trisynaptic loop 3 . The trisynaptic loop is implicated in the pathomechanism of hippocampal damage during SE.
In an animal model of SE, excitotoxic changes in hippocampal substructures were demonstrated. Pilocarpineinduced acute seizures strongly induce abnormal hippocampal neurogenesis. These abnormal changes are characterized by increased proliferation of neural progenitor cells and abnormal integration of the newly generated granular cells in the subgranular zone (SGZ) of the DG 4,5 . Aberrant integration caused by the newly generated neurons is termed mossy fiber sprouting. The mossy fibers extend into the hilus and project to excitatory (mossy cells) and inhibitory interneurons. Then, the fibers pass through the stratum lucidum and synapses of CA3 pyramidal neurons 6 . In the pilocarpine model, mossy fiber sprouting due to spontaneous seizures disrupts the synapses at CA3 and induces loss of cells in the CA3 and CA1 pyramidal cell layers. In brief, abnormal neurogenesis in the DG and aberrant neural network formation results in sequential neural degeneration in CA3 and CA1. www.nature.com/scientificreports/ Several new antiepileptic drugs have been introduced in recent years 7 ; however, most of them only have anticonvulsant effects; neuroprotective effects are very limited. Antiepileptic drugs fail to control seizures in 20-30% of patients 8,9 . Therefore, new alternatives to control epilepsy development are required. Photobiomodulation (PBM) therapy, previously known as low-level laser (light) therapy (LLLT) 10 , is a novel therapy for the treatment of various neurological disorders. Emerging evidence suggests that PBM protects neurons in epilepsy models, including an SE model [11][12][13][14] . In addition, PBM therapy causes hippocampal alterations 15 . However, the effects of this novel therapeutic approach on the recovery process are poorly understood. Based on previous studies that reported increased stem cell and progenitor cell proliferation with PBM therapy 16 , its effects on DG neurogenesis, and on CA3 and CA1, should be elucidated. Furthermore, recent studies have suggested that the functional connectivity among hippocampal substructures is highly complex, and not limited to a one-way loop (e.g., there is a direct connection between the entorhinal cortex and CA3 or CA1). Therefore, PBM may not reverse the pathomechanism underlying the excitotoxicity caused by SE.
In the present study, we administered PBM therapy to the depilated head of an SE animal model, allowing a portion of the light energy to reach the hippocampus. The population of mature neurons in DG was compared between the PBM therapy and SE only groups. In addition, the population of premature cells and their morphological changes were compared to evaluate the process of neurogenesis in detail. Finally, we evaluated the effect of PBM on sequential neuronal degeneration in CA3 and CA1.

Results
Hilar interneuron cell population in the DG in an SE animal model with or without PBM. Mice were injected with pilocarpine (320 mg/kg) to induce SE with a minimal mortality rate. The loss and neurogenesis of hilar interneurons are key events in the seizure-like discharges that cause further neuronal damage 17 . We evaluated the population of neuronal cells in this anatomical area 21 days after pilocarpine injection. Patients were treated with PBM for 1 (single, 36 J) or 5 (multiple, 180 J) consecutive days following 4 h of pilocarpine injection (Fig. 1D). Compared to the SE only group, the SE + 180 J PBM group showed higher population of NeuN, a mature neuronal marker (Fig. 2). However, there was no difference in the NeuN population between the SE only and SE + 36 J PBM groups (data not shown). The difference between SE only and SE + 180 J PBM  Proliferating cell/neuron population at the hilus in the DG of an SE animal model with or without PBM. It is important to identify the newly generated/proliferating cells in the DG because these cells are produced through neurogenesis, which could be responsible for the cascade of events that trigger excitotoxicity or the regeneration process required for recovery. Ki-67 is a marker of cell division, and Ki-67-positive cells were counted at three time points in this study (i.e., 7, 14, and 21 days after SE) (  (Fig. 4C). However, at 7 and 21 days, no difference was observed between the groups. Taken together, these results suggest that at all three time points after SE, newly generated/proliferating neurons (NeuN + /BrdU +) and non-neuronal cells (e.g., undifferentiated cells and glial cells) were observed in the DG. At higher doses of PBM, the number of proliferating cells was increased at 7 and 14 days. At 14 days, neuronal proliferation (NeuN + /BrdU +) was increased; however, at 7 days, total cell proliferation (Ki-67 + cells) was increased without an increase in neuronal proliferation (NeuN + /BrdU +). Therefore, neurogenesis likely occurs between 7 and 14 days after PBM. Detailed analysis of the undifferentiated and progenitor cell (early neuronal) populations and morphologies at these time points is necessary.

Undifferentiated/early neuronal cell population in the hilus in the DG of an SE animal model with or without PBM.
To determine whether PBM influences the growth and maturation of new cells in the DG, we performed immunohistochemical staining using anti-DCX, a microtubule-associated phosphoprotein expressed by the vast majority of BrdU + cells, and by cells co-expressing early neuronal antigens, but not by antigens specific to glia or undifferentiated cells 18 . At 7 days after PBM exposure, the intensity and number of DCX + cells in the DG did not change compared to SE alone (Fig. 5A). Similarly, at 21 days after PBM treatment, the number of DCX + cells and staining intensity did not differ compared to SE alone. However, at 14 days after 180 J PBM treatment, the DCX staining intensity was increased by 229.9 ± 22.5% compared to SE alone  www.nature.com/scientificreports/ tion on the GCL in response to PBM during maturation, we evaluated the distance of DCX + cells from the GCL. Overall, the migrated distance gradually increased from 7 to 14 and 21 days in the PBM 180 J-treated groups (Fig. 6A Neuronal cell population in CA3 and CA1 in an SE animal model with or without PBM. After abnormal neurogenesis, which begins in the DG, degeneration of other substructures of the hippocampus, such as CA3 and CA1, occurs. In the present study, we observed a mature neuronal population in CA3 and CA1 after SE, with or without PBM, at 21 days. As expected, neuronal loss was observed in CA3 and CA1 in the SE animal model (Fig. 7). The cellular continuity of mature neurons was incomplete in CA3 and CA1. On the other hand, in both PBM groups, the cellular continuity of mature neurons was relatively well-established in both CA3 and CA1.

Discussion
Based on the evidence that PBM promotes cell proliferation and differentiation, and that abnormal neurogenesis occurs in the hippocampus after SE, we hypothesized that PBM might play a modulatory role in abnormal hippocampal neurogenesis after SE. We found that PBM transformed SE-induced abnormal neurogenesis to normal neurogenesis. Therefore, PBM is an important modulator of neurogenesis in the hippocampus after SE, which may aid understanding of post-SE hippocampal pathological changes and the development of a potential therapeutic approach for SE. Hilar interneurons guide the maturation and integration of early neuronal cells, and are one of the primary cell types that degenerate during SE 19,20 . Initially, we measured the cell survival of hilar interneurons after SE and found that higher-energy irradiation of PBM increased NeuN-positive hilar interneuron cell survival at 21 days. This increase in the neuronal population could be due to the robust neurogenesis induced by PBM. Previous studies indicated that PBM can increase neurogenesis by stimulating the proliferation and differentiation of The ectopic migration of early neuronal cells is a key feature of SE-induced neurogenesis 4 . In the DG of adult rodents, most seizure-related early neuronal cells showed ectopic migration and did not integrate into the hilus or migrate toward the hilar/CA3 border 23,24 . In the present study, we found increased migration of early neuronal cells to the GCL after PBM compared to SE alone at all time points (i.e., 7, 14, and 21). The increased migration of early neuronal cells to the appropriate location (i.e., GCL) by PBM suggests that aberrant sprouting can be minimized by preventing neuronal differentiation and/or axon sprouting at improper locations. This study demonstrated that the neurogenesis-modulating effect of PBM protected the loop synapses to CA1 and CA3 (particularly CA1), such that the neurons were preserved on day 21 after SE.
In the present study, the neuroprotective effect of PBM was significant in CA1, but not CA3. Although the reason for this difference is not clear, there are several possible reasons. One of them, there may be a direct pathway between DG and CA1, outside of the already known loop synaptic pathway. Therefore, PBM may have a robust influence on the previously known synapse pathway from DG to CA1 via the entorhinal cortex 3 .
Many studies have reported improvement in cognitive function in various neurodegenerative diseases after PBM [25][26][27] ; therefore, PBM irradiation may also improve behavioral and cognitive function in the SE model. In www.nature.com/scientificreports/ the present study, the behavioral test (shown in our supplementary data S1) investigated whether PBM alleviated the symptoms of depression or anxiety induced by SE. There were result showing limited improvement only in high energy PBM. However, further studies with more detailed analysis of animal behavior regarding cognitive function is necessary. PBM irradiation may cause scalp heating 28 ; however, the thermal effects of PBM were not evaluated in this study. The photothermal effects of PBM are less likely to occur in brain tissue due to poor heat penetration into the brain 29 . In addition, we speculate that the heat at the skin and scalp did not modulate the therapeutic effect of PBM due to cytochrome c oxidase and cerebral hemodynamics 28 . However, the thermal effect should be evaluated in future studies, which should include histopathological examinations of the rat brain, particularly the hippocampus, as well as Nissl staining to monitor neuronal damage, TUNEL assay for assessing apoptosis, propidium iodide staining for analyzing necrosis, and electron microscopy to examine mitochondrial morphology. Finally, the synergistic anticonvulsive effects of adjunctive PBM with antiepileptic drugs should be evaluated in future studies. To allow the use of PBM therapy in epilepsy patients, studies in human cells should be performed.
In the present study, we demonstrated that PBM plays a key role in normal hippocampal neurogenesis, and that normal neurogenesis induced by PBM prevents SE-induced hippocampal neuronal loss. Thus, PBM is a novel approach to prevent seizure-induced neuronal degeneration, for which light devices may be developed in the future.  PBM treatment. PBM irradiation was performed using an 830-nm diode laser ( Fig. 1A; Wontech, Daejeon, South Korea) that penetrated the scalp and skull to reach the brain in the SE mouse model. PBM was anatomically irradiated to the scalp just above the hippocampus. As a result of measuring the laser transmittance for the scalp and skull, 63.7% of the original energy of the laser light reaches the neocortex area under the scalp 32 . During laser treatment, mice were immobilized using a restrainer and irradiated with PBM after 4 h of SE (Fig. 1B). Mice did not show any symptoms of depression or anxiety due to restraint (Fig. S1). The two PBM treatment groups were irradiated on a single and 5 consecutive days, respectively. The distance between the scalp and fiber end was 5 cm, the power density was 50 mW/cm 2 (maintained for 12 min), and the total radiant energy at the brain (spread over the entire head) was 22.9 J/cm 2 (Fig. 1C). The light source power density was measured using a laser power meter (PD-300 and VEGA power meter; Ophir, Darmstadt, Germany). The laser specifications are described in detail in Table 1.

Treatment parameters
Irradiance power at skin (mW/cm 2 ) 50 Exposure duration (s) 720 Radiant exposure at skin (J/cm 2 ) 36 Laser transmission ratio to skin and skull (%) 63.7 Irradiance power at brain (mW/cm 2 ) 31.8 Distance to the head from the end of a fiber (cm) 5 www.nature.com/scientificreports/ to denature the DNA. After rinsing in PBS, they were incubated with the primary anti-BrdU antibodies. After treatment with primary antibodies, the tissues were incubated with secondary antibodies conjugated with Alexa Fluor 488-conjugated anti-rabbit IgG (A11008; ThermoFisher Scientific, Waltham, MA, USA) and Alexa Fluor 555-conjugated anti-mouse IgG (A21422; ThermoFisher Scientific) for 1.5 h at room temperature. Then, the sections were incubated with secondary antibodies and stained with DAPI (D9542, 1:1,000; Sigma) for 10 min to visualize the nuclei. The immunostained tissues were mounted with Vectashield antifade mounting medium (H-1000; Vector Laboratories Inc., Burlingame, CA, USA). Alexa Fluor 488-(excitation, 488 nm; emission, 520 nm) and Alexa Fluor 555 (excitation, 561 nm; emission, 568 nm)-labeled tissues were imaged using a confocal microscope (FV3000; Olympus, Tokyo, Japan). Brain tissue was quantified on the basis of six 18-µm-thick coronal sections. A Z-stack depth of 15 μm was used, with 1-μm intervals. At least five individual brain samples per group were analyzed and three slices per brain samples were stained. Data are presented as mean number of cells/section ± standard error of mean (SEM). The researcher conducting the confocal imaging and the assessor performing the analysis of immunohistochemistry were blinded to the group assignment.
Statistical analysis. The data are presented as mean ± SEM. Statistical analysis was performed using oneway analysis of variance (ANOVA) followed by the Bonferroni test. The analyses were performed using Graph-Pad Prism version 7.04 (GraphPad Software, La Jolla, CA, USA). P-values < 0.05 were considered significant.

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.